Identification of eukaryotic growth-related genes and promoter isolation vector and method of use

ABSTRACT

A polynucleotide encoding chitin synthase (CHS1), an enzyme essential for cell wall synthesis and yeast cell growth, is provided. A maltose responsive promoter (MRP) isolated using the promoter library of the invention is also described. The present invention also provides a vector for isolation of a eukaryotic regulatory polynucleotide, i.e., promoter. The vector is useful in the method of the invention which comprises identifying a eukaryotic regulatory polynucleotide, i.e., promoter region, by complementing the growth of an auxotrophic host cell containing the vector of the invention, which includes a promoter region operably linked to a promoterless auxotrophic gene. The vector is introduced into the host cell chromosome by targeted integration. Also provided is a library containing host cells having the vector of the invention integrated in the chromosome of the host cell.

RELATED APPLICATION INFORMATION

This application is a divisional of application Ser. No. 09/004,225,filed Jan. 8, 1998 now U.S. Pat No. 6,020,133, which is a divisional ofapplication Ser. No. 08/551,437, filed Nov. 1, 1995 now U.S. Pat. No.5,824,545.

FIELD OF THE INVENTION

This invention relates generally to the field of gene expression andspecifically to genes essential for growth and to a vector and a methodfor the identification of such genes, as well as identification ofeukaryotic promoters.

BACKGROUND OF THE INVENTION

Many eukaryotic genes are regulated in an inducible, cell type-specificor constitutive manner. There are several types of structural elementswhich are involved in the regulation of gene expression. There arecis-acting elements, located in the proximity of, or within, genes whichserve to bind sequence-specific DNA binding proteins, as well astrans-acting factors. The binding of proteins to DNA is responsible forthe initiation, maintenance, or down-regulation of transcription ofgenes.

The cis-acting elements which control genes are called promoters,enhancers or silencers. Promoters are positioned next to the start siteof transcription and function in an orientation-dependent manner, whileenhancer and silencer elements, which modulate the activity ofpromoters, are flexible with respect to their orientation and distancefrom the start site of transcription.

For many years, various drugs have been tested for their ability toalter the expression of genes or the translation of their messages intoprotein products. One problem with existing drug therapy is that ittends to act indiscriminately on genes and promoters and thereforeaffects healthy cells as well as neoplastic cells. Likewise, in the caseof a pathogen-associated disease, it is critical to administer apathogen-specific therapy to avoid any detrimental effect on thenon-infected cells.

Chitin, a linear β-1,4 linked polymer of N-acetylglucosamine, is presentin the cell walls of all true fungi, but is absent from mammalian cells.Studies in S. cerevisiae (reviewed in Bulawa, C., Mol. Cell. Biol.12:1764, 1992; Cabib et al., Arch. Med. Res., 24:301, 1993) have shownthat the synthesis of chitin is surprisingly complex, requiring at leastthree isozymes encoded by the CHS1, CHS2, and CSD2 genes. In cell-freeextracts, all of the isozymes catalyze the formation of chitin usingUDP-N-acetylglucosamine as the substrate. In cells, each isozyme makeschitin at a unique location in the cell during a specified interval ofthe cell cycle. Genetic analyses indicate that CHS2 is involved in thesynthesis of the chitin-rich primary septum that separates mother anddaughter cells, CSD2 is required for synthesis of the chitin rings, andCHS1 plays a role in cell wall repair. Thus, the three isozymes are notfunctionally redundant and do not substitute for one another.

Chitin synthase genes have been identified from a diverse group offungi, and analysis of the deduced amino acid sequences of these geneshas lead to the identification of two chitin synthase gene families(Bowen, et al., Proc. Natl. Acad. Sci., USA, 89:519, 1992). Members ofone family are related to the S. cerevisiae CHS genes (CHS family).Based on sequence analyses, the CHS family can be subdivided intoclasses I, II, and III. Members of the second family are related to theS. cerevisiae CSD2 gene.

The functions of class II CHS genes have been investigated in a numberof fungi by gene disruption. In S. cerevisiae, the class II CHS mutant(designated chs2) is defective in cell separation (Bulawa and Osmond,Proc. Natl. Acad. Sci., USA, 87:7424, 1990; Shaw et al., J. Cell Biol.,114(1):111, 1990). In A. nidulans (Yanai et al., Biosci. 58(10):1828,1994) and U. maydis (Gold and Kronstad, Molecular Microbiology,11(5):897, 1994), class II CHS mutants (designated chsA and chs1,respectively) have no obvious phenotype. Thus, all of the class II CHSgenes studied to date are nonessential for growth. In addition, Young,et al. identified chitin synthase gene which encodes only part of thechitin synthase activity in C. albicans (Molec. Micro., 4(2):197, 1990).

There have been methods designed to identify virulence genes ofmicroorganisms involved in pathogenesis. For example, Osbourn, et al.utilized a promoter-probe plasmid for use in identifying promoters thatare induced in vivo in plants by Xanthomonas campestris (EMBO, J. 6:23,1987). Random chromosomal DNA fragments were cloned into a site in frontof a promoterless chloramphenicol acetyltransferase gene contained inthe plasmid and the plasmids were transferred into Xanthomonas to form alibrary. Individual transconjugates were introduced intochloramphenicol-treated seedlings to determine whether thetransconjugate displayed resistance to chloramphenicol in the plant.

Knapp, et al., disclosed a method for identifying virulence genes basedon their coordinate expression with other known virulence genes underdefined laboratory conditions (J. Bacteriol., 170:5059, 1988). Mahan, etal., (U.S. Pat. No. 5,434,065) described an in vivo genetic system toselect for microbial genes that are specifically induced when microbesinfect their host. The method depends on complementing the growth of anauxotrophic or antibiotic sensitive microorganism by integrating anexpression vector by way of homologous recombination into theauxotrophic or antibiotic sensitive microorganism's chromosome andinducing the expression of a synthetic operon which encodes transcripts,the expression of which are easily monitored in vitro following in vivocomplementation.

These systems all describe methods of identifying genes involved inpathogenesis in bacterial-host systems. There is a need to identifyspecific targets of eukaryotic pathogens, e.g., fungi, in an infectedcell which are associated with the expression of genes whose expressionproducts are implicated in disease, in order to increase efficacy oftreatment of infected cells and to increase the efficiency of developingdrugs effective against genes essential for survival of these pathogens.

The present invention provides a method for identifying targetsessential for growth as well as specific targets identified by themethod.

SUMMARY OF THE INVENTION

The present invention provides a yeast chitin synthase (CHS1)polypeptide and a polynucleotide encoding the polypeptide. In thepresent invention,the class II CHS gene of C. albicans (encoded by theCHS1 gene) is shown to be essential for growth under laboratoryconditions and for colonization of tissues during infection in vivo.Thus, CHS1 is a target for the development of antifungal drugs.

CHS1 inhibitors are useful for inhibiting the growth of a yeast. SuchCHS1 inhibitory reagents include, e.g., anti-CHS1 antibodies and CHS1antisense molecules.

CHS1 can be used to determine whether a compound affects (e.g.,inhibits) CHS1 activity, by incubating the compound with CHS1polypeptide, or with a recombinant cell expressing CHS1, underconditions sufficient to allow the components to interact, and thendetermining the effect of the compound on CHS1 activity or expression.

The invention also provides a vector for identifying a eukaryoticregulatory polynucleotide, including a selectable marker gene; arestriction endonuclease site located at the 5′ terminus of theselectable marker gene where a regulatory polynucleotide can be insertedto be operably linked to the selectable marker gene; and apolynucleotide for targeted integration of the vector into thechromosome of a susceptible host. Preferably, the eukaryotic regulatorypolynucleotide is a promoter region, and most preferably, a promoterregion of pathogenic yeast such as Candida albicans. The vector of theinvention is preferably transferred to a library of host cells, whereineach host cell contains the vector.

The vector of the invention can be used to identify a eukaryoticregulatory polynucleotide. The method involves inserting genomic DNA ofa eukaryotic organism into the vector, wherein the DNA is in operablelinkage with the selectable marker gene; transforming a susceptible hostwith the vector; detecting expression of the selectable marker gene,wherein expression is indicative of operable linkage to a regulatorypolynucleotide; and identifying the regulatory polynucleotide.

The vector of the invention also can be used to identify a compositionwhich affects the regulatory DNA (promoter). The method involvesincubating the composition to be tested and the promoter, underconditions sufficient to allow the promoter-containing vector of theinvention and the composition to interact, and then measuring the effectthe composition has on the promoter. The observed effect on the promotermay be either inhibitory or stimulatory.

The method of the invention is useful for identification of promotersfrom any eukaryote. Particularly preferred eukaryotes are fungalpathogens including, but not limited to, Candida albicans, Rhodotorulasp., Saccharomyces cerevisiae, Blastoschizomyces capitatus, Histoplasmacapsulatum, Aspergillus fumigatus, Coccidioides immitis,Paracoccidioides brasiliensis, Blastomyces dermatitidis, andCryptococcus neoformans.

The invention also features a regulatory polynucleotide (a promoter)isolated using a library of host cells containing the vector of theinvention; the promoter is a maltose responsive promoter (MRP), which isinduced by maltose and repressed by glucose. MRP is useful fordetermining whether a polynucleotide encodes a growth-associatedpolypeptide; the method involves incubating a cell containing thepolynucleotide operably linked with the MRP, under conditions whichrepress the regulatory polynucleotide, and then determining the effectof the expression of the polynucleotide on the growth of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a comparison of CHS1 clones.

FIGS. 1b-g is the nucleotide and deduced amino acid sequence of ChitinSynthase (CHS1) isolated from Candida albicans (SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:3).

FIG. 2a is a restriction map of the vector pBluescripts® II KS (+/−).

FIG. 2b is a restriction map of the vector pVGCA2.

FIGS. 3a-b are the nucleotide sequence of the maltose responsivepromoter (MRP) from C. albicans (SEQ ID NO:4).

FIG. 4 is a schematic illustration showing regulated expression of CHS1operatively linked to MRP.

FIG. 5 is a schematic illustration showing the bidirectional regulationcapability of MRP.

FIG. 6 is a restriction map of the pKW044 vector including the CHS1gene.

FIGS. 7A-D are a demonstration of gene inactivation during infection byMRP. Panels A and B show neutropenic and Panels C and D showimmunocompetent mice infected with the indicated strains of C. albicans.

DETAILED DESCRIPTION

The invention provides genes essential for growth, such as the chitinsynthase gene from Candida albicans (CaCHS1), as well as vectors foridentification of eukaryotic promoters. Preferably, the vector is usedfor the identification of promoters of fungal pathogens such as Candidaalbicans. The vectors allow identification of promoters and genes underthe control of such promoters, many of which are involved in theinfection process. A maltose responsive promoter (MRP) is provided as anexample of a promoter isolated using the vector of the invention.

Identification of a Yeast Gene Essential for Cell Growth

The invention provides a substantially pure chitin synthase (CHS1)polypeptide. The term “substantially pure” as used herein refers to CHS1which is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify CHS1 using standard techniques for proteinpurification. The substantially pure polypeptide will yield a singlemajor band on a non-reducing polyacrylamide gel. The purity of the CHS1polypeptide can also be determined by amino-terminal amino acid sequenceanalysis. CHS1 polypeptide includes functional fragments of thepolypeptide, provided that the activity of CHS1 remains. Smallerpeptides containing the biological activity of CHS1 are also included inthe invention.

The invention also provides polynucleotides encoding the CHS1 protein.These polynucleotides include DNA, cDNA and RNA sequences which encodeCHS1. It is understood that all polynucleotides encoding all or aportion of CHS1 are also included herein, as long as they encode apolypeptide with CHS1 activity. Such polynucleotides include naturallyoccurring, synthetic, and manipulated polynucleotides. For example, CHS1polynucleotide may be subjected to site-directed mutagenesis.

The polynucleotide sequence for CHS1 can be used to produce antisensesequences as well as sequences that are degenerate as a result of thedegeneracy of the genetic code; there are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention, provided the aminoacid sequence of CHS1 polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

Specifically disclosed herein is the yeast CHS1 gene, more specifically,the Candida albicans CHS1 gene. The sequence is 3084 base pairs long andcontains an open reading frame encoding a polypeptide 1027 amino acidsin length and having a molecular weight of about 116 kD as determined byreducing SDS-PAGE.

Preferably, the C. albicans CHS1 nucleotide sequence is SEQ ID NO:1 andthe deduced amino acid sequence is SEQ ID NO:2 (FIGS. 1b-g).

The polynucleotide encoding CHS1 includes SEQ ID NO:1 as well as nucleicacid sequences capable of hybridizing to SEQ ID NO:1 under stringentconditions. A complementary sequence may include an antisensenucleotide. When the sequence is RNA, the deoxynucleotides A, G, C, andT of SEQ ID NO:1 are replaced by ribonucleotides A, G, C, and U,respectively. Also included in the invention are fragments of theabove-described nucleic acid sequences that are at least 15 bases inlength, which is sufficient to permit the fragment to selectivelyhybridize to DNA that encodes the protein of SEQ ID NO:2 under stringentphysiological conditions.

The CHS1 polypeptide of the invention can be used to produce antibodieswhich are immunoreactive with or which specifically bind to epitopes ofthe CHS1 polypeptide. As used herein, the term “epitope” means anyantigenic determinant of an antigen to which an antibody to the antigenbinds.

Antibodies can be made to the protein of the invention, includingmonoclonal antibodies, which are made by methods well known in the art(Kohler, et al., Nature, 256:495, 1975; Current Protocols in MolecularBiology, Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain the ability to selectively bind with its antigen or receptor andare defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)2, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Methods of making thesefragments are known in the art. (See for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1988), incorporated herein by reference).

Antibodies which bind to the CHS1 polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide used to immunize an animal can be derived fromtranscribed/translated cDNA or chemical synthesis, and can be conjugatedto a carrier protein, if desired. Such commonly used carriers which canbe chemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is used to immunize the animal (e.g., a mouse, arat, or a rabbit).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

The invention also provides a method for inhibiting the growth of yeast,by contacting the yeast with a reagent which suppresses CHS1 activity.Preferably the yeast is C. albicans.

Where a disease or disorder is associated with the production of CHS1(e.g., a yeast infection), nucleic acid sequences that interfere withCHS1 expression at the translational level can be used to treat theinfection. This approach utilizes, for example, antisense nucleic acids,ribozymes, or triplex agents to block transcription or translation ofCHS1 mRNA, either by masking that mRNA with an antisense nucleic acid ortriplex agent or by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,as the cell will not translate a mRNA that is double-stranded. Antisenseoligomers of about 15 nucleotides are preferred, since they are easilysynthesized and are less likely to cause problems than larger moleculeswhen introduced into the CHS1-producing cell (e.g., a Candida albicans).The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (Marcus-Sakura, Anal.Biochemn., 172:289,1988).

Use of an oligonucleotide to block transcription is known as the triplexstrategy; the oligomer winds around double-helical DNA, forming athree-strand helix. These triplex compounds can be designed to recognizea unique site on a chosen gene (Maher, et al., Antisense Res. and Dev.,1(3):227, 1991; Helene, C., Anticancer Drug Design, 6(6):569, 1991).

The reagent used for inhibition of the growth of yeast by suppression ofCHS1 activity can be an anti-CHS1 antibody. Addition of such an antibodyto a cell or tissue suspected of containing a yeast, such as C.albicans, can prevent cell growth by inhibiting cell wall formation.

The invention also provides a method for detecting a yeast cell in ahost tissue, for example, which comprises contacting an anti-CHS1antibody or CHS1 polynucleotide with a cell having a yeast-associatedinfection and detecting binding to the antibody or hybridizing with thepolynucleotide, respectively. The antibody or polynucleotide reactivewith CHS1 or DNA encoding CHS1 is labeled with a label which allowsdetection of binding or hybridization to CHS1 or the DNA. An antibodyspecific for CHS1 polypeptide or polynucleotide specific for CHS1polynucleotide may be used to detect the level of CHS1 in biologicalfluids and tissues of a patient.

The antibodies of the invention can be used, for example, inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier.

The anti-CHS1 antibodies of the invention can be bound to a solidsupport and used to detect the presence of an antigen of the invention.Examples of well-known supports include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding antibodies, or will be able to ascertain such, using routineexperimentation.

The CHS1 antibodies of the invention can be used in vitro and in vivo tomonitor the course of amelioration of a yeast-associated disease in asubject. Thus, for example, by measuring the increase or decrease in thenumber of cells expressing antigen comprising CHS1 polypeptide of theinvention or changes in the concentration of such antigen present invarious body fluids, it is possible to determine whether a particulartherapeutic regimen aimed at ameliorating the yeast-associated diseaseis effective. The term “ameliorate” denotes a lessening of thedetrimental effect of the yeast-associated disease in the subjectreceiving therapy.

The CHS1 of the invention is also useful in a screening method toidentify compounds or compositions which affect the activity of theprotein. To determine whether a compound affects CHS1 activity, thecompound is incubated with CHS1 polypeptide, or with a recombinant cellexpressing CHS1, under conditions sufficient to allow the components tointeract; the effect of the compound on CHS1 activity or expression isthen determined.

The increase or decrease of chitin synthase transcription/translationcan be measured by adding a radioactive compound to the mixture ofcomponents, such as ³²P-ATP or ³⁵S-Met, and observing radioactiveincorporation into CHS1 transcripts or protein, respectively.Alternatively, other labels may be used to determine the effect of acomposition on CHS1 transcription/translation. For example, aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme could be used.Those of ordinary skill in the art will know of other suitable labels orwill be able to ascertain such, using routine experimentation. Analysisof the effect of a compound on CHS1 is performed by standard methods inthe art, such as Northern blot analysis (to measure gene expression)orSDS-PAGE (to measure protein product), for example. Further, CHS1enzymatic activity can also be determined, for example, by incorporationof labeled precursor of chitin. Preferably, such precursor isUDP-N-acetylglucoseamine.

Vector for Identification of a Eukaryotic Regulatory Polynucleotide

The vector contains at least one promoterless selectable marker gene anda restriction endonuclease cloning site located at the 5′ terminus ofthe selectable marker. A pool of chromosomal DNA fragments from aeukaryotic organism is inserted at the restriction endonuclease cloningsite in operable linkage with the selectable marker polynucleotide. Inaddition, the vector contains a polynucleotide sequence for targetedintegration of the vector into the chromosome of a susceptible host.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid, to which it has beenoperatively linked, from one genetic environment to another.

The term “regulatory polynucleotide” as used herein preferably refers toa promoter, but can also include enhancer elements. The vectors of theinvention contain a promoterless selectable marker gene having a cloningsite at the 5′ terminus of the gene. The vectors also include a cloningsite 5′ of the selectable marker gene, which is operably associated witha promoter. The term “operably associated” or “operably linked” refersto functional linkage between the promoter sequence and the controllednucleic acid sequence; the sequence and promoter are typicallycovalently joined, preferably by conventional phosphodiester bonds.

The expression vectors of the invention employ a promoterless gene forselection of a promoter sequence. The vectors contain other elementstypical of vectors, including an origin of replication, as well as geneswhich are capable of providing phenotypic selection of transformedcells. The transformed host cells can be grown in the appropriate mediaand environment, e.g., in fermentors, and cultured according totechniques known in the art to achieve optimal cell growth. The vectorsof the present invention can be expressed in vivo in either prokaryotesor eukaryotes. Methods of expressing DNA sequences containing eukaryoticcoding sequences in prokaryotes are well known in the art. Biologicallyfunctional plasmid DNA vectors used to incorporate DNA sequences of theinvention for expression and replication in the host cell are describedherein. For example, DNA can be inserted into yeast cells using thevectors of the invention. Various shuttle vectors for the expression offoreign genes in yeast have been reported (Heinemann, et al., Nature,340:205, 1989; Rose, et al., Gene, 60:237, 1987).

Host cells include microbial, yeast, and mammalian cells, e.g.,prokaryotes and eukaryotes such as yeast, filamentous fungi, and plantand animal cells.

Transformation or transfection with recombinant DNA may be carried outby conventional techniques well known to those skilled in the art. Wherethe host is prokaryotic, such as E. coli, competent cells which arecapable of DNA uptake can be prepared from cells harvested after theexponential growth phase and subsequently treated, i.e., by the CaCl₂method using procedures well known in the art.

Where the host cell is eukaryotic, various methods of DNA transfer canbe used. These include transfection of DNA by calciumphosphate-precipitates, conventional mechanical procedures such asmicroinjection, insertion of a plasmid encased in liposomes,spheroplast, electroporation, salt mediated transformation ofunicellular organisms, or the use of viral vectors. A library of hostcells, wherein each host cell contains a vector according to thedescription above, is also included in the invention.

Eukaryotic DNA can be cloned into prokaryotes using vectors well knownin the art. Because there are many functions in eukaryotic cells whichare absent in prokaryotes, (e.g., localization of ATP-generating systemsto mitochondria, association of DNA with histone, mitosis and meiosis,and differentiation of cells), the genetic control of such functionsmust be assessed in a eukaryotic environment. Many eukaryotic vectors,though, are capable of replication in E. coli, which is important foramplification of the vector DNA. Thus, vectors preferably containmarkers, e.g., LEU 2, HIS 3, URA 3, that can be selected easily inyeast, and in addition, also carry antibiotic resistance markers for usein E. coli. The selectable marker gene, which lies immediatelydownstream from the cloning site, preferably encodes a biosyntheticpathway enzyme of a eukaryote which relies on the enzyme for growth orsurvival. This biosynthetic pathway gene, once activated, willcomplement the growth of an auxotrophic host, deficient for the samebiosynthetic pathway gene in which it is integrated. Typically, genesencoding amino acid biosynthetic enzymes are utilized, since manystrains are available having at least one of these mutations, andtransformation events are easily selected by omitting the amino acidfrom the medium. Examples of markers include but are not limited toURA3, URA3-hisG, LEU2, LYS2, HIS3, HIS4, TRP1, ARG4, Hgm^(R), andTUN^(R). Preferably, the vector includes a promoterless URA3 gene.Expression of the C. albicans URA 3 gene is required for the infectionprocess, thus creating a strong selection pressure for those sequencescloned upstream of the promoterless URA3 gene that will be inducedduring the infection process.

The vector of the invention preferably includes a prokaryotic origin ofreplication or replicon, i.e., a DNA sequence having the ability todirect autonomous replication and maintenance of the recombinant DNAmolecule extra-chromosomally in a transformed prokaryotic host cell.Such origins of replication are well known in the art; preferred originsof replication are those that are efficient in the host organism, e.g.,the preferred host cell, E. coli. For vectors used in E. coli, apreferred origin of replication is ColE1, which is found in pBR322 and avariety of other common plasmids. Also preferred is the p15A origin ofreplication found on pACYC and its derivatives. The ColE1 and p15Areplicon have been extensively utilized in molecular biology, areavailable on a variety of plasmids, and are described, e.g., inSambrook, et al., Molecular Cloning: a Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, 1989).

The ColE1 and p15A replicons are particularly preferred for use in theinvention because they each have the ability to direct the replicationof a plasmid in E. coli while the other replicon is present in a secondplasmid in the same E. coli cell. In other words, ColE1 and p15A arenon-interfering replicons that allow the maintenance of two plasmids inthe same host (see, for example, Sambrook, et al., supra, at pages1.3-1.4).

The vector of the invention includes a polylinker multiple cloning sitefor insertion of selectable marker genes. A sequence of nucleotidesadapted for directional ligation, i.e., a polylinker, is a region of theDNA expression vector that (1) operatively links for replication andtransport the upstream and downstream translatable DNA sequences, and(2) provides a site for directional ligation of a DNA sequence into thevector. Typically, a directional polylinker is a sequence of nucleotidesthat defines two or more restriction endonuclease recognition sequences.Upon restriction cleavage, the two sites yield cohesive termini to whicha translatable DNA sequence can be ligated to the DNA expression vector.Preferably, the two restriction sites provide, upon restrictioncleavage, cohesive termini that are non-complementary and thereby permitdirectional insertion of a translatable DNA sequence into the cassette.Where the sequence of nucleotides adapted for directional ligationdefines numerous restriction sites, it is referred to as a multiplecloning site.

Additionally, the vector may contain a phenotypically selectable markergene to identify host cells which contain the expression vector.Examples of markers typically used in prokaryotic expression vectorsinclude antibiotic resistance genes for ampicillin (β-lactamases),tetracycline and chloramphenicol (chloramphenicol acetyltransferase).

The vector contains a polynucleotide sequence for targeted integrationof the vector into the chromosome of a susceptible host. Targetedintegration, as opposed to random integration, results in more stabletransformants and avoids position effects or integration into genesrequired for growth and infection. Preferably, the gene for targetedintegration is also a selectable marker, thereby allowing theidentification of transformants that contain the vector. Such genesinclude the adenine biosynthesis (ADE2) gene of Candida albicans. Asusceptible host is a host having a site recognized by thepolynucleotide of the vector for targeted integration.

Promoters identified by the method of the invention can be inducible orconstitutive promoters. Inducible promoters can be regulated, forexample, by nutrients (e.g., carbon sources, nitrogen sources, andothers), drugs (e.g., drug resistance), environmental agents that arespecific for the infection process (e.g., serum response), andtemperature (e.g., heat shock, cold shock).

Identification of a Eukaryotic Regulatory Polynucleotide

The selection method of the invention utilizes an auxotrophic organism,or an organism that has a mutation in a biosynthetic pathway geneencoding a functional biosynthetic enzyme necessary for the growth ofthe organism. When a functional or wild-type copy of a biosyntheticpathway gene is inserted into an auxotroph, the expression of thewild-type biosynthetic pathway gene provides the auxotroph with thebiosynthetic enzyme required for growth or survival. The process ofreplacing a missing or non-functional gene of an auxotroph with afunctional homologous gene in order to restore the auxotroph's abilityto survive within a host cell is called “complementation”.

Complementation of the auxotroph, according to the present invention, isaccomplished by construction of a vector having a promoterlessstructural gene encoding a—biosynthetic enzyme, i.e., a selectablemarker polynucleotide, as described above. The cloning site for thepromoter of interest is at the 5′ terminus of the structural geneencoding the biosynthetic enzyme. Consequently, a promoter regionoperatively linked to any gene or set of genes will control theexpression of that gene or genes. In order to be controlled by thepromoter, the gene must be positioned downstream from the promoter.

The structural gene encoding a biosynthetic enzyme in the vector of theinvention does not contain recognition sequences for regulatory factorsto allow transcription of the structural gene. Consequently, theproduct(s) encoded by the structural gene is not capable of beingexpressed unless a promoter sequence is inserted into the cloning site5′ to the structural gene.

A second structural gene in the vector allows for targeted insertion andintegration into the host cell's chromosomal DNA. Optionally, the vectormay contain additional genes, such as those encoding selective markersfor selection in bacteria. Typically drug resistance genes such as thosedescribed above are used for such selection.

In the method of the invention, total genomic DNA is isolated from theorganism, e.g., Candida albicans, and then partially enzymaticallydigested, resulting in a pool of random chromosomal fragments. Thevector of the invention is cleaved at the restriction/cloning site, andmixed with the cleaved chromosomal DNA. The chromosomal fragments areligated into the vector to produce a library, i.e., each vector containsa random chromosomal fragment so that the pool of vectors isrepresentative of the entire organism's genome. The vectors containingthe chromosomal fragments are then introduced into the host organism(e.g., an auxotrophic strain or drug resistant strain of Candidaalbicans) by methods well know in the art. For example, the vectors maybe introduced by transformation.

After the vector is introduced into the host (e.g., auxotrophic), thevector may integrate into the auxotroph's chromosome by targetedintegration. This step can be detected by selection, as described above.For example, the preferred polynucleotide for targeted insertion andintegration in Candida albicans is the ADE2 gene. The presence of thisgene is detectable by growth of the organism on adenine deficient media.

The expression of the biosynthetic enzyme gene, e.g., URA3, whetherunder constitutive or inducible conditions, is identified bycomplementation of a host cell strain in which the gene is defective ormissing, e.g., URA3-. Only those host cells which can grow in mediumlacking the nutritional supplement, e.g., uracil, will be expected tocontain a cloned functional promoter sequence.

Identification of a Yeast Regulatory Polynucleotide Capable of Inductionand Repression

In another aspect, the invention provides an isolated regulatorypolynucleotide, the MRP promoter, characterized in that it is induced bymaltose and repressed by glucose. MRP of the invention is exemplified bythe nucleotide sequence of SEQ ID NO:4 (FIGS. 3a-b), wherein thesequence is 1734 base pairs in length. MRP was isolated from a promoterlibrary based on expression of the Ura3 gene of C. albicans as describedabove. MRP functions bidirectionally, that is, genes flanking MRP both5′ and 3′ are controlled by this regulatory polynucleotide.

The MRP of the invention is useful for identifying genes which areessential for cell growth. Thus, the invention provides a method fordetermining whether a polynucleotide encodes a growth-associatedpolypeptide, by incubating a cell containing the polynucleotide operablylinked with the MRP regulatory polynucleotide, under conditions whichrepress the regulatory polynucleotide, and determining the effect of thetested polynucleotide on the growth of the cell.

MRP of the invention promotes transcription in the presence of maltose,while the ability of MRP to promote transcription is repressed byglucose. A cell having a polynucleotide of interest operably linked toMRP can be grown on a glucose containing medium to determine whether thepolynucleotide of interest is essential for cell growth. MRP isrepressed on glucose, thus repressing transcription of the operablylinked polynucleotide, therefore, if a cell grown on a glucosecontaining-medium dies, the polynucleotide is determined to be essentialfor cell growth.

MRP can be used to induce (maltose) or repress (glucose) expression of agene operably linked to MRP. It is also envisioned that MRP may beuseful for decreasing the expression of a target gene operably linked toMRP, such that the cell containing the MRP-gene of interest is nowextremely sensitive to a compound of interest. For example, it may bedesirable to increase susceptibility or resistance to a particulartherapeutic compound. Similarly, MRP is useful for inducing expressionof a gene operatively linked to MRP, by growing a host cell containing aMRP-gene construct on a maltose-containing medium. It may be desirableto elevate gene expression for screening various therapeutic compoundsfor their effect on the gene product.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES Example 1

Isolation of Chitin Synthase from Candida albicans

Using Southern blotting, the restriction maps for the cloned CHS1 genecontained in pJAIV and the genomic CHS1 locus were produced, however,the maps were found not to match. Additional studies indicated thatpJAIV contained two nonadjacent genomic DNA fragments as diagrammed inFIG. 1a. As a consequence, pJAIV lacked the 5′ end of CHS1. To clonethis region, a plasmid rescue strategy was employed. Plasmid pKW025,which contains a 600 bp KpnI/EcoRI fragment of CHS1, and a 1.4 kbCandida URA3 gene cloned into pSK(−), was cut with ClaI and transformedinto Candida albicans strain CAI-4. Transformants were examined bySouthern blot and strain CAI-4A was identified, containing pKW025integrated at the CHSl locus. Genomic DNA was extracted from CA-1-4A andcut with Hind III. Because pKWO25 and the sequenced portion of CHSlcontain no Hind III sites, this digestion yields on a single DNAfragment pKW025 plus the genomic CHS1 locus with flanking regionsextending to the 5′ and 3′ Hind III sites. Ligation was carried out witha low DNA concentration to promote intramolecular ligation events, andthe DNA transformed into E. coli. Recovered plasmids were screened byPCR to verify that they contained contiguous CHS1 sequence.

Plasmid pKW030 (12 kb total) was identified and contained approximately2 kb of CHS1 sequence upstream of the XhoI site. A 3.6 kb HindIII/PstIfragment was cloned into the HindIII/PstI sites of pSK(−), formingplasmid pKW032. The 3′ region of the gene was derived from plasmidpKW013 (originally derived from pJA-IV). A 3.5 kb BstEII/−NotI fragmentwas cloned into the BstEII/NotI sites of pKW032, forming plasmid pKW035.pKW035 was cut with various restriction enzymes, and Southern blotanalysis also carried out to confirm that the insert was indeed anuninterrupted CHS1 gene whose restriction pattern matched that of thechromosomal CHS1.

The insert was sequenced by standard methods and the nucleotide anddeduced amino acid sequence are shown in FIG. 1b-g (SEQ ID NO:1 and 2).

EXAMPLE 2 Construction of Promoter Isolation Vector

The Candida albicans URA3 gene was amplified by PCR and a Sail site wasinserted next to the ATG. The 3′ primer used contained a genomic XbaIsite. The SalI/XbaI fragment was cloned in Bluescript KS+ at SalI/XbaI.The C. albicans EcoRV genomic fragment containing the ADE2 gene wascloned in the above plasmid at the XhoI site of the Bluescriptpolylinker.

The Ca URA3 gene was amplified by PCR using the following primers:

5′ Primer URA3-ATG: 5′-GGAGGA[GTCGAC]ATGACAGTCAACAC-3′ (SEQ ID NO:5)            SalI 3′ Primer URA3-XbaI: 5′-CGCATTAAAGC[TCTAGA]AGAACCACC-3′(SEQ ID NO:6)                 XbaI (Underlined regions: genomic)

The PCR reaction was as follows:

100 ng DNA, 50 pmoles each primer, 2.5 mM dNTP, 2.5 mM Mg Cl₂₁, 0.5U TaqPolymerase/100 μl.

Reaction

step 1: 2 min 94° C.

step 2: 1 min 94° C.

step 3: 1 min 57° C.

step 4: 11/2 min 72° C.

step 5: steps 2-4×30 times

step 6: 10 min 72° C.

step 7: Hold 4° C.

For the cloning, 20 μl of the PCR reaction was run on 0.7% low meltingagarose gel and the band was purified using the Promega (Madison, Wis.)PCR purification resin. The purified band and 1 μg of StrategeneKS+bluescript (FIG. 2a; Stratagene, La Jolla, Calif.) were digested withSalI and XbaI, gel isolated (as above) and eluted in 50 μl water.

The ligation reaction was performed as follows: Ligation (20 μl): 1 μlvector, 10 μl digested PCR band, 2 μl T4 ligase buffer, 1 μl (2 units)T4 ligase (Boehrringer), 6 μl H₂O, over night at room temperature. 10 μlof the ligation was used to transform Strategene XL1 Blueultra-competent cells selecting for ampicillin resistance. Individualcolonies were grown in LB+ampicillin and plasmid DNA was isolated usingthe Quiagen (Chatsworth, Calif.) spin columns.

The above plasmid was digested with XhoI, filled in with Klenow for 30min and dephosporylated. with acid phosphatase for 5 min. The band wasgel purified as above. The EcoRV fragment containing the Ca ADE2 genewas cloned into the plasmid using the conditions described above (FIG.2b).

EXAMPLE 3 Isolation and Characterization of a Maltose Induced/GlucoseRepressed Promoter of C. albicans

Using the promoter probe vector pVGCAV2 (based on URA3 expression), alibrary was constructed which inserted 1-2 kb Sau3A fragments (isolatedby sucrose gradient centrifugation) upstream (5′) of the promoterlessURA3 reporter gene into the vector. The vector plasmid was cut with SalIand partially end filled with dT and dC while the insert fragments(Sau3A cut) were partially filled in with dG and dA. These partial fillin reactions left 2 bp overhangs that are compatible for a ligationreaction. The results of the ligation of the library were introducedinto E. coli strain DH5A by electroporation, and gave rise to 76,500independent transformants. Sixteen randomly picked colonies all provedto have inserts indicating the library was sound.

The plasmid library was extracted from E. coli by standard plasmidisolation procedures and cut at the unique BamHI site within the ADE2gene for targeted integration of the ADE locus of C. albicans strainCaI8 (ade2ura3). The ade2 mutation of CaI8 allows for selection oftransformants and the ura3 mutation of CaI8 permits monitoring ofexpression of the reporter gene URA3. A first pool of 10,000 independentCaI8 transformants was tested for regulated URA3 expression. The CaI8transformants were plated on Synthetic Dextrose [glucose medium (2%glucose (w/v) and yeast nitrogen base without amino acids at 6.7 g/L(Difco)) without uridine] to determine the frequency of transformantsexpressing the URA3 gene constitutively. Fourteen per cent of theCandida CaI8 transformants expressed varying levels of the URA3 gene asdetermined by the ability to form colonies on a medium lacking uridinesupplementation. The pool was then treated with the compound 5-FOA toremove these transformants expressing the URA3 gene constitutively(transformants expressing URA3 convert 5-Fluoro-orotic acid to a toxiccompound and thus can be eliminated from the pool). To isolate promotersresponding to specific carbon sources, aliquots of the pool were grownon synthetic glucose medium supplemented with uridine and replicated tosynthetic maltose medium without uridine. Candida transformants able toproduce colonies on the unsupplemented maltose medium putativelycontained a maltose inducible promoter. Four strains (MRP-2, MRP-5,MRP-6, MRP-7) were shown to show maltose dependent growth that wasrepressed upon the addition of glucose.

Chromosomal DNA was extracted from the Candida CaI8 transformantsexhibiting maltose dependent growth (MRP strains) and digested with therestriction enzyme BamHI to “release the MRP clones.” The “released”plasmids were ligated and introduced into E. coli by transformation.These E. coli transformants were used as a source of plasmid DNA fordideoxy/chain termination sequencing. Initial sequencing data using aprimer to URA3 sequences just downstream of the insert (3′) indicatedall the MRP strains contained the same insert. Sequencing data obtainedusing a primer to ADE2 sequences (5′ to the insert DNA with respect toURA3 transcription indicated the clone contained part of a maltase geneand regulatory sequences (FIGS. 3a-b, SEQ ID NO:4). The entire sequenceof the clone was assembled and the portion of the maltase ORF containedon the insert was shown to be approximately 70% sequence identical to apreviously cloned promoter of C. albicans maltase (CAMAL2) (Geber, etal., J. Bacteriology, 174:6992, 1992).

EXAMPLE 4 Identification of Genes Essential for Yeast Cell Growth

This experiment used the MRP promoter as a gene disruption tool, and theC. albicans CHS1 gene. A strain was constructed and designated KWC340,in which CHS1 expression is regulated by the carbon source present inthe growth medium. Transcription of CHS1 was induced by maltose andrepressed by glucose. In maltose containing medium, KWC340 grows at thesame rate as a wild-type strain. When KWC340 is transferred toglucose-containing medium, cells stop growing and eventually die. Threegenerations after transfer to glucose, short chains of cells grow butfail to separate. Ten generations after transfer, growth has stopped.Long chains and clumps of cells are seen; a large percentage of thecells are anucleate or multinucleate, indicating a defect in nuclearsegregation. Viability is reduced approximately 500-fold relative to acontrol culture, as judged by plating efficiency.

As a first step in constructing a strain in which the sole functionalCHS1 gene was under the control of the MRP fragment, a vector wasconstructed in pKS termed KWO44 with the following features (see FIGS.4-6):

(a) the plasmid contained URA3 for selection of transformants in theUra-strains CaI4 (CHS1/CHS1) and 167b (CHS1/chs1::hisG)

(b) a 1088 bp PCR fragment of the MRP sequence (see attached FIG. 5showing sites of PCR primers)

(c) 1479 bp of the C. albicans CHS1 N-terminus that contains a uniqueXhoI site to target the transformation/-integration event.

This construct fuses the ATG initiation codon of the CHS1 gene at thesame position as the URA3 gene (original reporter gene used to isolatethe MRP clone) with respect to the MRP fragment. Integration of thisconstruct at the remaining wild-type CHS1 allele in strain 167b placesthe sole functional CHS1 gene under the control of the transcriptionalcontrol of the MRP fragment. After transformation this type ofintegrants were recovered as confirmed by Southern analysis. Theseintegrants grew well on maltose containing medium (inducing conditions)but died when replicated to glucose containing medium.

When injected into mice, the MRP-CHS1 integrants were avirulent;thesymptoms diagnostic of candidiasis were not observed, and the kidneysfrom the mice were sterile. Thus CHS1 is essential for growth in vitroand in vivo. Briefly, ICR 4-week-old male mice (Harlan Sprague Dawley)were housed five per cage; food and water were given ad libitumaccording to the National Institutes of Health guidelines for theethical treatment of animals. Strains of C. albicans were grown in SMmedium [2% maltose, 0.7% yeast nitrogen base without amino acids (DifcoLaboratories, Detroit, Mich.)] to a density of 10⁷ cells/ml. Cells wereharvested, washed, resuspended in sterile water, and injected into mice(10⁶ cells/immunocompetent mouse, 10⁴ cells/neutropenic mouse) via thelateral tail veins. For each strain of C. albicans, five mice wereinfected. Cages were checked three times daily for mice dead or moribund(exhibiting severe lethargy, vertigo, and ruffled fur) mice. Moribundmice were euthenized by cervical dislocation and necropsied. The leftand right kidneys were removed and examined for colonization by C.albicans. In experiments using neutropenic mice, cyclophosphamide wasadministered (150 mg/kg) by intraperitoneal injection 96 and 24 hoursprior to infection. Injections were repeated every three days for theduration of the experiment. Neutropenia was verified by comparing thepercentage of neutrophils to total number of leukocytes before and afterinjection with cyclophosphamide.

FIG. 7, panels A-D, shows the results of the in vivo experiment.Neutropenic (panels A & B) and immunocompetent (panels C & D) mice wereinfected with the indicated strains of C. albicans: clinical isolate(strain SC5314, , panels A & C); MRP::URA3 (strain MRP2, a derivative ofSC5314 containing one copy of URA3 which is regulated by MRP, □, panelsA & C); MRP::CHS1 (strain KWC340, a derivative of SC5314 containing onecopy of CHS1 which is regulated by MRP, A, panels B & D); andCHS1/MRP::CHS1 (strain KWC352, a derivative of SC5314 containing twocopies of CHS1; one regulated by MRP, the other by the CHS1 promoter, o,panels B & D).

In conclusion, these results show the MRP clone controls the expressionof two non cognate genes (CHS1 and URA3) in a regulated manner anddemonstrate the utility of the MRP sequence as a genetic tool in C.albicans for target validation (determination of gene essentiallity).

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

6 3084 base pairs nucleic acid double linear not provided CodingSequence 1...3081 1 ATG AAG AAT CCA TTT GAC AGT GGC AGT GAC GAT GAA GATCCA TTT CTT 48 Met Lys Asn Pro Phe Asp Ser Gly Ser Asp Asp Glu Asp ProPhe Leu 1 5 10 15 AGT AAT CCA CAA TCT GCA CCA TCA ATG CCC TAC GCA GCATAT TTC CCA 96 Ser Asn Pro Gln Ser Ala Pro Ser Met Pro Tyr Ala Ala TyrPhe Pro 20 25 30 CTG TCG ACT AGT GGA TCT CCA TTT CAC CAA CAG CAA TCC CCAAGA CAA 144 Leu Ser Thr Ser Gly Ser Pro Phe His Gln Gln Gln Ser Pro ArgGln 35 40 45 TCA CCT AAT ATT TTT TCC AGA AGT ACT GCA AGA GCA ACT AGT GACAGA 192 Ser Pro Asn Ile Phe Ser Arg Ser Thr Ala Arg Ala Thr Ser Asp Arg50 55 60 ACA TCG CCC CGC AAG ACA TAC CAA CCA TTG AAT TTT GAC AGT GAG GAC240 Thr Ser Pro Arg Lys Thr Tyr Gln Pro Leu Asn Phe Asp Ser Glu Asp 6570 75 80 GAA GAT GCT AAA GAA AGC GAA TTT ATG GCT GCA ACC TCA AAG CTG AAT288 Glu Asp Ala Lys Glu Ser Glu Phe Met Ala Ala Thr Ser Lys Leu Asn 8590 95 ATG AGC ATA TAT GAT AAT ACC CCG AAC TTA CAA TTC AAC AAA AGC GGC336 Met Ser Ile Tyr Asp Asn Thr Pro Asn Leu Gln Phe Asn Lys Ser Gly 100105 110 GCA GCC ACA CCA AGA GCA CAA TTC ACA TCG AAA GAA TCT CCG AAA AGA384 Ala Ala Thr Pro Arg Ala Gln Phe Thr Ser Lys Glu Ser Pro Lys Arg 115120 125 CAA AAA ACT ACT GAA GTG ACC ATT GAC TTT GAC AAT GAT GAT GAT AAC432 Gln Lys Thr Thr Glu Val Thr Ile Asp Phe Asp Asn Asp Asp Asp Asn 130135 140 AAT CAC ACC TTA GAA TTT GAA AAT GGG TCA CCT CGT CGT TCA TTT CGT480 Asn His Thr Leu Glu Phe Glu Asn Gly Ser Pro Arg Arg Ser Phe Arg 145150 155 160 AGT AGT GCT ATA AGC AGC GAA AGA TTT TTG CCT CCT CCA CAA CCAATT 528 Ser Ser Ala Ile Ser Ser Glu Arg Phe Leu Pro Pro Pro Gln Pro Ile165 170 175 TTC TCT CGA GAA ACA TTT GCT GAA GCC AAC TCC CGT GAA GAA GAAAAA 576 Phe Ser Arg Glu Thr Phe Ala Glu Ala Asn Ser Arg Glu Glu Glu Lys180 185 190 TCG GCA GAT CAA GAA ACA TTA GAT GAA AAA TAC GAT TAT GAT TCATAC 624 Ser Ala Asp Gln Glu Thr Leu Asp Glu Lys Tyr Asp Tyr Asp Ser Tyr195 200 205 CAG AAG GGT TAT GAG GAA GTA GAA ACA TTG CAT TCG GAA GGT ACAGCT 672 Gln Lys Gly Tyr Glu Glu Val Glu Thr Leu His Ser Glu Gly Thr Ala210 215 220 TAT AGT GGC TCA TCT TAT TTG TCG GAT GAT GCC AGT CCT GAA ACTACA 720 Tyr Ser Gly Ser Ser Tyr Leu Ser Asp Asp Ala Ser Pro Glu Thr Thr225 230 235 240 GAT TAC TTT GGA GCT TCA ATT GAT GGT AAT ATT ATG CAC AACATT AAC 768 Asp Tyr Phe Gly Ala Ser Ile Asp Gly Asn Ile Met His Asn IleAsn 245 250 255 AAT GGA TAC GTA CCA AAT AGA GAA AAA ACC ATT ACC AAA AGAAAA GTG 816 Asn Gly Tyr Val Pro Asn Arg Glu Lys Thr Ile Thr Lys Arg LysVal 260 265 270 AGA TTA GTT GGT GGC AAA GCA GGT AAC TTG GTC TTG GAG AATCCA GTT 864 Arg Leu Val Gly Gly Lys Ala Gly Asn Leu Val Leu Glu Asn ProVal 275 280 285 CCA ACA GAG TTG AGA AAA GTG TTG ACC AGA ACC GAG TCT CCATTT GGT 912 Pro Thr Glu Leu Arg Lys Val Leu Thr Arg Thr Glu Ser Pro PheGly 290 295 300 GAG TTT ACC AAC ATG ACA TAC ACA GCG TGC ACT TCG CAG CCAGAT ACT 960 Glu Phe Thr Asn Met Thr Tyr Thr Ala Cys Thr Ser Gln Pro AspThr 305 310 315 320 TTT TCT GCT GAA GGG TTC ACC TTA AGA GCT GCC AAA TACGGC AGA GAA 1008 Phe Ser Ala Glu Gly Phe Thr Leu Arg Ala Ala Lys Tyr GlyArg Glu 325 330 335 ACT GAG ATT GTC ATT TGT ATA ACC ATG TAT AAT GAG GACGAA GTT GCA 1056 Thr Glu Ile Val Ile Cys Ile Thr Met Tyr Asn Glu Asp GluVal Ala 340 345 350 TTT GCC AGA ACT ATG CAT GGT GTG ATG AAA AAT ATC GCTCAT TTG TGC 1104 Phe Ala Arg Thr Met His Gly Val Met Lys Asn Ile Ala HisLeu Cys 355 360 365 TCA CGC CAT AAA TCC AAA ATA TGG GGC AAA GAT AGC TGGAAA AAA GTT 1152 Ser Arg His Lys Ser Lys Ile Trp Gly Lys Asp Ser Trp LysLys Val 370 375 380 CAA GTG ATA ATT GTT GCA GAT GGT AGA AAT AAA GTT CAACAA TCC GTT 1200 Gln Val Ile Ile Val Ala Asp Gly Arg Asn Lys Val Gln GlnSer Val 385 390 395 400 CTT GAA TTG CTT ACG GCA ACA GGC TGC TAT CAA GAAAAT TTG GCC AGG 1248 Leu Glu Leu Leu Thr Ala Thr Gly Cys Tyr Gln Glu AsnLeu Ala Arg 405 410 415 CCC TAT GTC AAC AAT AGC AAA GTA AAT GCC CAT TTGTTT GAA TAT ACC 1296 Pro Tyr Val Asn Asn Ser Lys Val Asn Ala His Leu PheGlu Tyr Thr 420 425 430 ACT CAA ATA TCT ATC GAT GAG AAC TTG AAA TTC AAAGGA GAT GAA AAA 1344 Thr Gln Ile Ser Ile Asp Glu Asn Leu Lys Phe Lys GlyAsp Glu Lys 435 440 445 AAC CTT GCA CCA GTT CAA GTC TTG TTC TGT TTG AAAGAA CTG AAC CAA 1392 Asn Leu Ala Pro Val Gln Val Leu Phe Cys Leu Lys GluLeu Asn Gln 450 455 460 AAG AAA ATC AAT TCC CAT AGA TGG CTT TTT AAT GCCTTT TGT CCT GTC 1440 Lys Lys Ile Asn Ser His Arg Trp Leu Phe Asn Ala PheCys Pro Val 465 470 475 480 TTG GAC CCC AAT GTT ATT GTT CTT TTA GAT GTGGGT ACC AAA CCC GAT 1488 Leu Asp Pro Asn Val Ile Val Leu Leu Asp Val GlyThr Lys Pro Asp 485 490 495 AAC CAT GCC ATT TAT AAT CTA TGG AAA GCA TTCGAT AGA GAT TCC AAT 1536 Asn His Ala Ile Tyr Asn Leu Trp Lys Ala Phe AspArg Asp Ser Asn 500 505 510 GTA GCA GGG GCT GCT GGT GAA ATT AAA GCG ATGAAA GGT AAA GGT TGG 1584 Val Ala Gly Ala Ala Gly Glu Ile Lys Ala Met LysGly Lys Gly Trp 515 520 525 ATT AAT CTT ACA AAT CCA TTA GTT GCG TCA CAGAAT TTT GAG TAT AAA 1632 Ile Asn Leu Thr Asn Pro Leu Val Ala Ser Gln AsnPhe Glu Tyr Lys 530 535 540 TTG TCC AAT ATT CTT GAT AAA CCG TTG GAA TCACTT TTT GGA TAC ATT 1680 Leu Ser Asn Ile Leu Asp Lys Pro Leu Glu Ser LeuPhe Gly Tyr Ile 545 550 555 560 TCT GTG TTA CCA GGT GCA TTG TCT GCA TATCGA TAC ATT GCC TTG AAA 1728 Ser Val Leu Pro Gly Ala Leu Ser Ala Tyr ArgTyr Ile Ala Leu Lys 565 570 575 AAC CAC GAT GAT GGT ACA GGG CCA TTG GCTTCT TAT TTC AAA GGT GAA 1776 Asn His Asp Asp Gly Thr Gly Pro Leu Ala SerTyr Phe Lys Gly Glu 580 585 590 GAT TTA CTC TGT TCA CAT GAC AAA GAC AAAGAG AAT ACC AAA GCT AAC 1824 Asp Leu Leu Cys Ser His Asp Lys Asp Lys GluAsn Thr Lys Ala Asn 595 600 605 TTT TTC GAA GCA AAT ATG TAC TTG GCT GAAGAC AGA ATC CTT TGT TGG 1872 Phe Phe Glu Ala Asn Met Tyr Leu Ala Glu AspArg Ile Leu Cys Trp 610 615 620 GAA TTG GTA TCA AAA AGA AAT GAC AAT TGGGTT CTT AAA TTT GTT AAA 1920 Glu Leu Val Ser Lys Arg Asn Asp Asn Trp ValLeu Lys Phe Val Lys 625 630 635 640 CTG GCA ACC GGT GAA ACT GAT GTT CCTGAA ACA ATT GCA GAA TTT CTT 1968 Leu Ala Thr Gly Glu Thr Asp Val Pro GluThr Ile Ala Glu Phe Leu 645 650 655 TCG CAA AGA CGA AGA TGG ATT AAT GGTGCC TTT TTT GCT GCT TTG TAC 2016 Ser Gln Arg Arg Arg Trp Ile Asn Gly AlaPhe Phe Ala Ala Leu Tyr 660 665 670 TCC TTG TAT CAC TTT AGA AAA ATA TGGACG ACT GAC CAT TCG TAT GCT 2064 Ser Leu Tyr His Phe Arg Lys Ile Trp ThrThr Asp His Ser Tyr Ala 675 680 685 AGA AAA TTT TGG CTA CAT GTC GAA GAATTC ATT TAT CAA TTG GTA TCA 2112 Arg Lys Phe Trp Leu His Val Glu Glu PheIle Tyr Gln Leu Val Ser 690 695 700 TTA TTG TTT TCA TTT TTT TCT TTG AGTAAT TTC TAT TTA ACA TTT TAT 2160 Leu Leu Phe Ser Phe Phe Ser Leu Ser AsnPhe Tyr Leu Thr Phe Tyr 705 710 715 720 TTT TTG ACA GGT TCA TTG GTG TCTTAC AAA AGT CTT GGT AAA AAA GGT 2208 Phe Leu Thr Gly Ser Leu Val Ser TyrLys Ser Leu Gly Lys Lys Gly 725 730 735 GGA TTT TGG ATT TTC ACA TTA TTCAAT TAT CTC TGT ATC GGT GTT TTG 2256 Gly Phe Trp Ile Phe Thr Leu Phe AsnTyr Leu Cys Ile Gly Val Leu 740 745 750 ACA TCT TTG TTC ATT GTC TCC ATTGGT AAT AGA CCA CAT GCA TCA AAG 2304 Thr Ser Leu Phe Ile Val Ser Ile GlyAsn Arg Pro His Ala Ser Lys 755 760 765 AAT ATT TTC AAA ACA TTA ATC ATATTG TTA ACC ATA TGT GCA TTA TAC 2352 Asn Ile Phe Lys Thr Leu Ile Ile LeuLeu Thr Ile Cys Ala Leu Tyr 770 775 780 GCA TTG GTG GTT GGA TTT GTG TTTGTT ATC AAT ACT ATT GCT ACT TTT 2400 Ala Leu Val Val Gly Phe Val Phe ValIle Asn Thr Ile Ala Thr Phe 785 790 795 800 GGA ACC GGT GGA ACA TCT ACCTAT GTG CTC GTT AGT ATT GTG GTT TCA 2448 Gly Thr Gly Gly Thr Ser Thr TyrVal Leu Val Ser Ile Val Val Ser 805 810 815 TTG TTG TCC ACC TAT GGT CTTTAT ACG TTA ATG TCC ATT TTG TAC TTG 2496 Leu Leu Ser Thr Tyr Gly Leu TyrThr Leu Met Ser Ile Leu Tyr Leu 820 825 830 GAC CCA TGG CAC ATG TTG ACTTGT TCT GTA CAA TAC TTT TTG ATG ATT 2544 Asp Pro Trp His Met Leu Thr CysSer Val Gln Tyr Phe Leu Met Ile 835 840 845 CCA TCG TAC ACT TGT ACA TTACAA ATA TTT GCA TTT TGT AAT ACT CAC 2592 Pro Ser Tyr Thr Cys Thr Leu GlnIle Phe Ala Phe Cys Asn Thr His 850 855 860 GAT GTC TCG TGG GGT ACA AAAGGT GAC AAC AAT CCA AAA GAA GAT TTG 2640 Asp Val Ser Trp Gly Thr Lys GlyAsp Asn Asn Pro Lys Glu Asp Leu 865 870 875 880 AGT AAT CAG TAC ATT ATTGAG AAA AAT GCC AGT GGA GAA TTT GAG GCT 2688 Ser Asn Gln Tyr Ile Ile GluLys Asn Ala Ser Gly Glu Phe Glu Ala 885 890 895 GTT ATT GTT GAT ACA AATATC GAT GAA GAT TAC CTT GAG ACA TTA TAT 2736 Val Ile Val Asp Thr Asn IleAsp Glu Asp Tyr Leu Glu Thr Leu Tyr 900 905 910 AAT ATC AGG TCA AAG AGATCA AAC AAA AAA GTG GCT TTG GGC CAT TCT 2784 Asn Ile Arg Ser Lys Arg SerAsn Lys Lys Val Ala Leu Gly His Ser 915 920 925 GAA AAG ACG CCT CTT GATGGT GAT GAT TAT GCA AAA GAC GTT CGT ACT 2832 Glu Lys Thr Pro Leu Asp GlyAsp Asp Tyr Ala Lys Asp Val Arg Thr 930 935 940 AGA GTT GTG TTG TTT TGGATG ATT GCA AAT TTG GTA TTT ATA ATG ACC 2880 Arg Val Val Leu Phe Trp MetIle Ala Asn Leu Val Phe Ile Met Thr 945 950 955 960 ATG GTA CAA GTT TACGAG CCA GGT GAT ACC GGA AGA AAC ATT TAT TTG 2928 Met Val Gln Val Tyr GluPro Gly Asp Thr Gly Arg Asn Ile Tyr Leu 965 970 975 GCC TTT ATT TTG TGGGCA GTG GCA GTG TTG GCT CTT GTC AGA GCT ATT 2976 Ala Phe Ile Leu Trp AlaVal Ala Val Leu Ala Leu Val Arg Ala Ile 980 985 990 GGC TCT CTT GGA TACTTG ATA CAA ACA TAT GCA CGG TTT TTT GTG GAA 3024 Gly Ser Leu Gly Tyr LeuIle Gln Thr Tyr Ala Arg Phe Phe Val Glu 995 1000 1005 TCG AAG AGT AAATGG ATG AAA CGA GGA TAT ACC GCG CCG AGT CAC AAT 3072 Ser Lys Ser Lys TrpMet Lys Arg Gly Tyr Thr Ala Pro Ser His Asn 1010 1015 1020 CCA TTA AATTAG 3084 Pro Leu Asn 1025 1027 amino acids amino acid linear proteininternal not provided 2 Met Lys Asn Pro Phe Asp Ser Gly Ser Asp Asp GluAsp Pro Phe Leu 1 5 10 15 Ser Asn Pro Gln Ser Ala Pro Ser Met Pro TyrAla Ala Tyr Phe Pro 20 25 30 Leu Ser Thr Ser Gly Ser Pro Phe His Gln GlnGln Ser Pro Arg Gln 35 40 45 Ser Pro Asn Ile Phe Ser Arg Ser Thr Ala ArgAla Thr Ser Asp Arg 50 55 60 Thr Ser Pro Arg Lys Thr Tyr Gln Pro Leu AsnPhe Asp Ser Glu Asp 65 70 75 80 Glu Asp Ala Lys Glu Ser Glu Phe Met AlaAla Thr Ser Lys Leu Asn 85 90 95 Met Ser Ile Tyr Asp Asn Thr Pro Asn LeuGln Phe Asn Lys Ser Gly 100 105 110 Ala Ala Thr Pro Arg Ala Gln Phe ThrSer Lys Glu Ser Pro Lys Arg 115 120 125 Gln Lys Thr Thr Glu Val Thr IleAsp Phe Asp Asn Asp Asp Asp Asn 130 135 140 Asn His Thr Leu Glu Phe GluAsn Gly Ser Pro Arg Arg Ser Phe Arg 145 150 155 160 Ser Ser Ala Ile SerSer Glu Arg Phe Leu Pro Pro Pro Gln Pro Ile 165 170 175 Phe Ser Arg GluThr Phe Ala Glu Ala Asn Ser Arg Glu Glu Glu Lys 180 185 190 Ser Ala AspGln Glu Thr Leu Asp Glu Lys Tyr Asp Tyr Asp Ser Tyr 195 200 205 Gln LysGly Tyr Glu Glu Val Glu Thr Leu His Ser Glu Gly Thr Ala 210 215 220 TyrSer Gly Ser Ser Tyr Leu Ser Asp Asp Ala Ser Pro Glu Thr Thr 225 230 235240 Asp Tyr Phe Gly Ala Ser Ile Asp Gly Asn Ile Met His Asn Ile Asn 245250 255 Asn Gly Tyr Val Pro Asn Arg Glu Lys Thr Ile Thr Lys Arg Lys Val260 265 270 Arg Leu Val Gly Gly Lys Ala Gly Asn Leu Val Leu Glu Asn ProVal 275 280 285 Pro Thr Glu Leu Arg Lys Val Leu Thr Arg Thr Glu Ser ProPhe Gly 290 295 300 Glu Phe Thr Asn Met Thr Tyr Thr Ala Cys Thr Ser GlnPro Asp Thr 305 310 315 320 Phe Ser Ala Glu Gly Phe Thr Leu Arg Ala AlaLys Tyr Gly Arg Glu 325 330 335 Thr Glu Ile Val Ile Cys Ile Thr Met TyrAsn Glu Asp Glu Val Ala 340 345 350 Phe Ala Arg Thr Met His Gly Val MetLys Asn Ile Ala His Leu Cys 355 360 365 Ser Arg His Lys Ser Lys Ile TrpGly Lys Asp Ser Trp Lys Lys Val 370 375 380 Gln Val Ile Ile Val Ala AspGly Arg Asn Lys Val Gln Gln Ser Val 385 390 395 400 Leu Glu Leu Leu ThrAla Thr Gly Cys Tyr Gln Glu Asn Leu Ala Arg 405 410 415 Pro Tyr Val AsnAsn Ser Lys Val Asn Ala His Leu Phe Glu Tyr Thr 420 425 430 Thr Gln IleSer Ile Asp Glu Asn Leu Lys Phe Lys Gly Asp Glu Lys 435 440 445 Asn LeuAla Pro Val Gln Val Leu Phe Cys Leu Lys Glu Leu Asn Gln 450 455 460 LysLys Ile Asn Ser His Arg Trp Leu Phe Asn Ala Phe Cys Pro Val 465 470 475480 Leu Asp Pro Asn Val Ile Val Leu Leu Asp Val Gly Thr Lys Pro Asp 485490 495 Asn His Ala Ile Tyr Asn Leu Trp Lys Ala Phe Asp Arg Asp Ser Asn500 505 510 Val Ala Gly Ala Ala Gly Glu Ile Lys Ala Met Lys Gly Lys GlyTrp 515 520 525 Ile Asn Leu Thr Asn Pro Leu Val Ala Ser Gln Asn Phe GluTyr Lys 530 535 540 Leu Ser Asn Ile Leu Asp Lys Pro Leu Glu Ser Leu PheGly Tyr Ile 545 550 555 560 Ser Val Leu Pro Gly Ala Leu Ser Ala Tyr ArgTyr Ile Ala Leu Lys 565 570 575 Asn His Asp Asp Gly Thr Gly Pro Leu AlaSer Tyr Phe Lys Gly Glu 580 585 590 Asp Leu Leu Cys Ser His Asp Lys AspLys Glu Asn Thr Lys Ala Asn 595 600 605 Phe Phe Glu Ala Asn Met Tyr LeuAla Glu Asp Arg Ile Leu Cys Trp 610 615 620 Glu Leu Val Ser Lys Arg AsnAsp Asn Trp Val Leu Lys Phe Val Lys 625 630 635 640 Leu Ala Thr Gly GluThr Asp Val Pro Glu Thr Ile Ala Glu Phe Leu 645 650 655 Ser Gln Arg ArgArg Trp Ile Asn Gly Ala Phe Phe Ala Ala Leu Tyr 660 665 670 Ser Leu TyrHis Phe Arg Lys Ile Trp Thr Thr Asp His Ser Tyr Ala 675 680 685 Arg LysPhe Trp Leu His Val Glu Glu Phe Ile Tyr Gln Leu Val Ser 690 695 700 LeuLeu Phe Ser Phe Phe Ser Leu Ser Asn Phe Tyr Leu Thr Phe Tyr 705 710 715720 Phe Leu Thr Gly Ser Leu Val Ser Tyr Lys Ser Leu Gly Lys Lys Gly 725730 735 Gly Phe Trp Ile Phe Thr Leu Phe Asn Tyr Leu Cys Ile Gly Val Leu740 745 750 Thr Ser Leu Phe Ile Val Ser Ile Gly Asn Arg Pro His Ala SerLys 755 760 765 Asn Ile Phe Lys Thr Leu Ile Ile Leu Leu Thr Ile Cys AlaLeu Tyr 770 775 780 Ala Leu Val Val Gly Phe Val Phe Val Ile Asn Thr IleAla Thr Phe 785 790 795 800 Gly Thr Gly Gly Thr Ser Thr Tyr Val Leu ValSer Ile Val Val Ser 805 810 815 Leu Leu Ser Thr Tyr Gly Leu Tyr Thr LeuMet Ser Ile Leu Tyr Leu 820 825 830 Asp Pro Trp His Met Leu Thr Cys SerVal Gln Tyr Phe Leu Met Ile 835 840 845 Pro Ser Tyr Thr Cys Thr Leu GlnIle Phe Ala Phe Cys Asn Thr His 850 855 860 Asp Val Ser Trp Gly Thr LysGly Asp Asn Asn Pro Lys Glu Asp Leu 865 870 875 880 Ser Asn Gln Tyr IleIle Glu Lys Asn Ala Ser Gly Glu Phe Glu Ala 885 890 895 Val Ile Val AspThr Asn Ile Asp Glu Asp Tyr Leu Glu Thr Leu Tyr 900 905 910 Asn Ile ArgSer Lys Arg Ser Asn Lys Lys Val Ala Leu Gly His Ser 915 920 925 Glu LysThr Pro Leu Asp Gly Asp Asp Tyr Ala Lys Asp Val Arg Thr 930 935 940 ArgVal Val Leu Phe Trp Met Ile Ala Asn Leu Val Phe Ile Met Thr 945 950 955960 Met Val Gln Val Tyr Glu Pro Gly Asp Thr Gly Arg Asn Ile Tyr Leu 965970 975 Ala Phe Ile Leu Trp Ala Val Ala Val Leu Ala Leu Val Arg Ala Ile980 985 990 Gly Ser Leu Gly Tyr Leu Ile Gln Thr Tyr Ala Arg Phe Phe ValGlu 995 1000 1005 Ser Lys Ser Lys Trp Met Lys Arg Gly Tyr Thr Ala ProSer His Asn 1010 1015 1020 Pro Leu Asn 1025 3084 base pairs nucleic aciddouble linear not provided 3 TACTTCTTAG GTAAACTGTC ACCGTCACTG CTACTTCTAGGTAAAGAATC ATTAGGTGTT 60 AGACGTGGTA GTTACGGGAT GCGTCGTATA TGATCACTGTCTTGTAGCGG GGCGTTCTGT 120 ATGGTTGGTA ACTTAAAACT GYCACTCCTG CTTCTACGATTTCTTTCGCT TAAATACCGA 180 AAGGGTGACA GCTGATCACC TAGAGGTAAA GTGGTTGTCGTTAGGGGTTC TGTTAGTGGA 240 TTATAAAAAA GGTCTTCATG ACGTTCTCGT CGTTGGAGTTTCGACTTATA CTCGTATATA 300 CTATTATGGG GCTTGAATGT TAAGTTGTTT TCGCCGCGTCGGTGTGGTTC TCGTGTTAAG 360 TGTAGCTTTC TTAGAGGCTT TTCTGTTTTT TGATGACTTCACTGGTAACT GAAACTGTTA 420 CTACTACTAT TGTTAGTGTG GAATCTTAAA CTTTTACCCAGTGGAGCAGC AAGTAAAGCA 480 TCATCACGAT ATTCGTCGCT TTCTAAAAAC GGAGGAGGTGTTGGTTAAAA GAGAGCTCTT 540 TGTAAACGAC TTCGGTTGAG GGCACTTCTT CTTTTTAGCCGTCTAGTTCT TTGTAATCTA 600 CTTTTTATGC TAATACTAAG TATGGTCTTC CCAATACTCCTTCATCTTTG TAACGTAAGC 660 CTTCCATGTC GAATATCACC GAGTAGAATA AACAGCCTACTACGGTCAGG ACTTTGATGT 720 CTAATGAAAC CTCGAAGTTA ACTACCATTA TAATACGTGTTGTAATTGTT ACCTATGCAT 780 GGTTTATCTC TTTTTTGGTA ATGGTTTTCT TTTCACTCTAATCAACCACC GTTTCGTCCA 840 TTGAACCAGA ACCTCTTAGG TCAAGGTTGT CTCAACTCTTTTCACAACTG GTCTTGGCTC 900 AGAGGTAAAC CACTCAAATG GTTGTACTGT ATGTGTCGCACGTGAAGCGT CGGTCTATGA 960 AAAAGACGAC TTCCCAAGTG GAATTCTCGA CGGTTTATGCCGTCTCTTTG ACTCTAACAG 1020 TAAACATATT GGTACATATT ACTCCTGCTT CAACGTAAACGGTCTTGATA CGTACCACAC 1080 TACTTTTTAT AGCGAGTAAA CACGAGTGCG GTATTTAGGTTTTATACCCC GTTTCTATCG 1140 ACCTTTTTTC AAGTTCACTA TTAACAACGT CTACCATCTTTATTTCAAGT TGTTAGGCAA 1200 GAACTTAACG AATGCCGTTG TCCGACGATA GTTCTTTTAAACCGGTCCGG GATACAGTTG 1260 TTATCGTTTC ATTTACGGGT AAACAAACTT ATATGGTGAGTTTATAGATA GCTACTCTTG 1320 AACTTTAAGT TTCCTCTACT TTTTTTGGAA CGTGGTCAAGTTCAGAACAA GACAAACTTT 1380 CTTGACTTGG TTTTCTTTTA GTTAAGGGTA TCTACCGAAAAATTACGGAA AACAGGACAG 1440 AACCTGGGGT TACAATAACA AGAAAATCTA CACCCATGGTTTGGGCTATT GGTACGGTAA 1500 ATATTAGATA CCTTTCGTAA GCTATCTCTA AGGTTACATCGTCCCCGACG ACCACTTTAA 1560 TTTCGCTACT TTCCATTTCC AACCTAATTA GAATGTTTAGGTAATCAACG CAGTGTCTTA 1620 AAACTCATAT TTAACAGGTT ATAAGAACTA TTTGGCAACCTTAGTGAAAA ACCTATGTAA 1680 AGACACAATG GTCCACGTAA CAGACGTATA GCTATGTAACGGAACTTTTT GGTGCTACTA 1740 CCATGTCCCG GTAACCGAAG AATAAAGTTT CCACTTCTAAATGAGACAAG TGTACTGTTT 1800 CTGTTTCTCT TATGGTTTCG ATTGAAAAAG CTTCGTTTATACATGAACCG ACTTCTGTCT 1860 TAGGAAACAA CCCTTAACCA TAGTTTTTCT TTACTGTTAACCCAAGAATT TAAACAATTT 1920 GACCGTTGGC CACTTTGACT ACAAGGACTT TGTTAACGTCTTAAAGAAAG CGTTTCTGCT 1980 TCTACCTAAT TACCACGGAA AAAACGACGA AACATGAGGAACATAGTGAA ATCTTTTTAT 2040 ACCTGCTGAC TGGTAAGCAT ACGATCTTTT AAAACCGATGTACAGCTTCT TAAGTAAATA 2100 GTTAACCATA GTAATAACAA AAGTAAAAAA AGAAACTCATTAAAGATAAA TTGTAAAATA 2160 AAAAACTGTC CAAGTAACCA CAGAATGTTT TCAGAACCATTTTTTCCACC TAAAACCTAA 2220 AAGTGTAATA AGTTAATAGA GACATAGCCA CAAAACTGTAGAAACAAGTA ACAGAGGTAA 2280 CCATTATCTG GTGTACGTAG TTTCTTATAA AAGTTTTGTAATTAGTATAA CAATTGGTAT 2340 ACACGTAATA TGCGTAACCA CCAACCTAAA CACAAACAATAGTTATGATA ACGATGAAAA 2400 CCTTGGCCAC CTTGTAGATG GATACACGAG CAATCATAACACCAAAGTAA CAACAGGTGG 2460 ATACCAGAAA TATGCAATTA CAGGTAAAAC ATGAACCTGGGTACCGTGTA CAACTGAACA 2520 AGACATGTTA TGAAAAACTA CTAAGGTAGC ATGTGAACATGTAATGTTTA TAAACGTAAA 2580 ACATTATGAG TGCTACAGAG CACCCCATGT TTTCCACTGTTGTTAGGTTT TCTTCTAAAC 2640 TCATTAGTCA TGTAATAACT CTTTTTACGG TCACCTCTTAAACTCCGACA ATAACAACTA 2700 TGTTTATAGC TACTTCTAAT GGAACTCTGT AATATATTATAGTCCAGTTT CTCTAGTTTG 2760 TTTTTTCACC GAAACCCGGT AAGACTTTTC TGCGGAGAACTACCACTACT AATACGTTTT 2820 CTGCAAGCAT GATCTCAACA CAACAAAACC TACTAACGTTTAAACCATAA ATATTACTGG 2880 TACCATGTTC AAATGCTCGG TCCACTATGG CCTTCTTTGTAAATAAACCG GAAATAAAAC 2940 ACCCGTCACC GTCACAACCG AGAACAGTCT CGATAACCGAGAGAACCTAT GAACTATGTT 3000 TGTATACGTG CCAAAAAACA CCTTAGCTTC TCATTTACCTACTTTGCTCC TATATGGCGC 3060 GGCTCAGTGT TAGGTAATTT AATC 3084 1734 basepairs nucleic acid single linear not provided 4 ATAATCGTTG TGCTACTGGTAGCTAGTTTC TGCTCTCTCA CTATANGGTC TTAGTGTTGA 60 CTGTCATGTC GATCAAGTTACTTACAGGTA AATTATTGAG TTTCAATAAG GTTGGTTTCG 120 TTGTGGCTAG TTTTTTCGATGTTTTACAAA ATGAAAAAAA ACTTAATACA TTTAAGCCAA 180 CAGCTTATTG TAGGTGCTCCTTTCATTATT CGTACTTCCT ACCCCATGGA GTTTAAAATG 240 ATAAYYGAAA TTTAAAGCCAACTAGCCAAC TAGCCAACTA GCCAGCTAGC MAGMCAAGAC 300 AAAACTAATC ACAAAGACTAAAAGAAAGTG TAGTTATAAA TCATTGCGAG AATTATTGCG 360 AAANGATATT CCGCTTTTCAAAAAAACATT ATTGCGAAAA TCATTGCNGA NGAAAGGGGG 420 AGTTATTTTT GGGGTACTACTATGCATGTG TTGTTGTCAA TGTCTACCAC AAAAAGGGGC 480 TTCTTTCAAT TGATAAACCTACCAAAACAT CTGGTAATCA AAAGCTACTT GTGTGAGACT 540 ATATTTATTG TAGATTACACCCCGCTCTAC AAAGTTACCA TGAAGACAAA ACAACTTGTT 600 TGAAGTTATA TGAATCGATGTTAAAAATCT GCGTCTCGTG GAGAGTAACT TGATTATGTT 660 AGGTCTGCTA TCGTTTATACTATGACCGCA TCATATACAG GACATTAGAG CATCCTAAAT 720 TAAATCATCC CATTGTTTCAAGTTTCTTTG TTTAGCAAAG AGACAGTTCC AACTTGTTGT 780 CGTCATAATT ATCGGAATAATTTAAGCGAG GAAAAGTTGT GAAACAAATT GAAGAGTGGA 840 GTGTGGGGGA GGGGGAGGGAAACAAGGAAG TATACCTCCA CCAAGTAGAA CCCAAATACT 900 CCACGTAATC AACAACAAGTAGCCATATAA TTCAAAATTT GTAGTAGTTG GGCAAATAAT 960 ATTTATACCC CCCCACTCCCCCAACCTTCC AATTTTCCTC TTCCTCTGGG AATTTTTTTT 1020 TTTGAAATAC AAATCTCTTTTAAAACCAAC TTAAACCTAT TAATTATGAC AATTGAATAT 1080 ACTTGGTGGA AAGACGCTACTATTTATCAA ATTTGGCCTG CTTCATATAA AGATTCCAAT 1140 GGTGATGGAA TTGGTGATATTCCAGGGATA ATTTCTACAT TAGATTATCT TAAAAATTTA 1200 GGAATTGATA TTATTTGGTTAAGTCCAATG TATAAATCCC CTATGGAAGA TATGGGTTAT 1260 GATATTAGTG ATTATGAATCTATAAATCCT GATTTTGGTA CTATGGAAGA CATGCAAAAT 1320 TTAATTGATG GATGTCATGAAAGAGGAATG AAAATTATTT GTGATTTAGT AGTTAATCAT 1380 ACATCATCTG AACATGAATGGTTTAAACAA TCAAGATCAC TGAAATCAAA CCCTAAAAGA 1440 GATTGGTATA TTTGGAAACCACCGAGAATT GACGCNAAAA ACTGGTGNAA AAATTACCAC 1500 CAAATAATTG GGGGTCATTTTTTTCAGGAT CAGCATGGGA TATGATGAAT TAACCGATGA 1560 ATATTATTTA AGATTATTTGCCAAGGGACA ACCTGATTTA AATTGGGAAA ATGAAGAAAG 1620 TCGTCAAGCA ATTTATAATTCTGCCATGAA ATCATGGTTT GATAAAGGTG TTGATGGATT 1680 TAGAATTGAT GTTGCTGGATNATATTCTAA AGATCGACCT CNGAATCAAA GGAA 1734 26 base pairs nucleic acidsingle linear not provided 5 GGAGGAGTCG ACATGACAGT CAACAC 26 26 basepairs nucleic acid single linear cDNA not provided 6 CGCATTAAAGCTCTAGAAGA ACCACC 26

What is claimed is:
 1. An isolated regulatory polynucleotide comprisingthe sequence of SEQ ID NO:4, characterized in that it is induced bymaltose and repressed by glucose.
 2. The regulatory polynucleotide ofclaim 1, wherein the regulatory polynucleotide is derived from a yeastcell.
 3. A method for determining whether a test polynucleotide encodesa growth-associated polypeptide, said method comprising: a) incubating afungal cell comprising the test polynucleotide operably linked with theregulatory polynucleotide of claim 1, under conditions which repress theregulatory polynucleotide; and b) determining the effect of the testpolynucleotide on the growth of the cell.
 4. The method of claim 3,wherein the effect is inhibition of cell growth.
 5. An isolatedregulatory polynucleotide of claim 1, further comprising a codingsequence operably linked to the isolated regulatory polynucleotide.
 6. Avector comprising an isolated regulatory polynucleotide of claim
 1. 7. Ahost cell comprising an isolated regulatory polynucleotide of claim 1.8. A host cell comprising a vector of claim 6.